1. Field of the Invention
The present invention relates generally to the screening of compound and combinatorial libraries. More particularly, the present invention relates to a method and apparatus for performing assays, particularly thermal shift assays.
2. Related Art
In recent years, pharmaceutical researchers have turned to combinatorial libraries as sources of new lead compounds for drug discovery. A combinatorial library is a collection of chemical compounds which have been generated, by either chemical synthesis or biological synthesis, by combining a number of chemical xe2x80x9cbuilding blocksxe2x80x9d as reagents. For example, a combinatorial polypeptide library is formed by combining a set of amino acids in every possible way for a given compound length (i.e., the number of amino acids in a polypeptide compound). Millions of chemical compounds can theoretically be synthesized through such combinatorial mixing of chemical building blocks. Indeed, one investigator has observed that the systematic, combinatorial mixing of 100 interchangeable chemical building blocks results in the theoretical synthesis of 100 million tetrameric compounds or 10 billion pentameric compounds (Gordon, E. M. et al., J. Med. Chem. 37:1233-1251 (1994)).
The rate of combinatorial library synthesis is accelerated by automating compound synthesis and evaluation. For example, DirectedDiversity(copyright) is a computer based, iterative process for generating chemical entities with defined physical, chemical and/or bioactive properties. The DirectedDiversity(copyright) system is disclosed in U.S. Pat. No. 5,463,564, which is herein incorporated by reference in its entirety.
Once a library has been constructed, it must be screened to identify compounds which possess some kind of biological or pharmacological activity. To screen a library of compounds, each compound in the library is equilibrated with a target molecule of interest, such as an enzyme. A variety of approaches have been used to screen combinatorial libraries for lead compounds. For example, in an encoded library, each compound in a chemical combinatorial library can be made so that an oligonucleotide xe2x80x9ctagxe2x80x9d is linked to it. A careful record is kept of the nucleic acid tag sequence for each compound. A compound which exerts an effect on the target enzyme is selected by amplifying its nucleic acid tag using the polymerase chain reaction (PCR). From the sequence of the tag, one can identify the compound (Brenner, S. et al., Proc. Natl. Acad. Sci. USA 89:5381-5383 (1992)). This approach, however, is very time consuming because it requires multiple rounds of oligonucleotide tag amplification and subsequent electrophoresis of the amplification products.
A filamentous phage display peptide library can be screened for binding to a biotinylated antibody, receptor or other binding protein. The bound phage is used to infect bacterial cells and the displayed determinant (i.e., the peptide ligand) is then identified (Scott, J. K. et al., Science 249:386-390 (1990)). This approach suffers from several drawbacks. It is time consuming. Peptides which are toxic to the phage or to the bacterium cannot be studied. Moreover, the researcher is limited to investigating peptide compounds.
In International Patent Application WO 94/05394 (1994), Hudson, D. et al., disclose a method and apparatus for synthesizing and screening a combinatorial library of biopolymers on a solid-phase plate, in an array of 4xc3x974 to 400xc3x97400. The library can be screened using a fluorescently labeled, radiolabeled, or enzyme-linked target molecule or receptor. The drawback to this approach is that the target molecule must be labeled before it can be used to screen the library.
A challenge presented by currently available combinatorial library screening technologies is that they provide no information about the relative binding affinities of different ligands for a receptor protein. This is true whether the process for generating a combinatorial library involves phage library display of peptides (Scott, J. K. et al., Science 249:386-390 (1990)), random synthetic peptide arrays (Lam, K. S. et al., Nature 354:82-84 (1991)), encoded chemical libraries (Brenner, S. et al., Proc. Natl. Acad. Sci. USA 89:5381-5383 (1992)), the method of Hudson (Intl. Appl. WO 94/05394), or most recently, combinatorial organic synthesis (Gordon, E. et al., J. Med. Chem. 37:1385-1399 (1994)).
To acquire quantitative binding data from the high throughput screening of ligand affinities for a target enzyme, researchers have relied on assays of enzyme activity. Enzymes lend themselves to high throughput screening because the effect of ligand binding can be monitored using kinetic assays. The experimental endpoint is usually a spectrophotometric change. Using a kinetic assay, most researchers use a two-step approach to lead compound discovery. First, a large library of compounds is screened against the target enzyme to determine if any of the library compounds are active. These assays are usually performed in a single concentration (between 10xe2x88x924-10xe2x88x926 M) with one to three replicates. Second, promising compounds obtained from the first screen (i.e., compounds which display activity greater than a predetermined value) are usually re-tested to determine a 50% inhibitory concentration (IC50), an inhibitor association constant (Ki), or a dissociation constant (Kd). This two-step approach, however, is very labor intensive, time-consuming and prone to error. Each retested sample must either be retrieved from the original assay plate or weighed out and solubilized again. A concentration curve must then be created for each sample and a separate set of assay plates must be created for each assay.
There are other problems associated with the biochemical approach to high throughput screening of combinatorial libraries. Typically, a given assay is not applicable to more than one receptor. That is, when a new receptor becomes available for testing, a new assay must be developed. For many receptors, reliable assays are simply not available. Even if an assay does exist, it may not lend itself to automation. Further, if a Ki is the endpoint to be measured in a kinetic assay, one must first guess at the concentration of inhibitor to use, perform the assay, and then perform additional assays using at least six different concentrations of inhibitor. If one guesses too low, an inhibitor will not exert its inhibitory effect at the suboptimal concentration tested.
In addition to the drawbacks to the kinetic screening approach described above, it is difficult to use the kinetic approach to identify and rank ligands that bind outside of the active site of the enzyme. Since ligands that bind outside of the active site do not prevent binding of spectrophotometric substrates, there is no spectrophotometric change to be monitored. An even more serious drawback to the kinetic screening approach is that non-enzyme receptors cannot be assayed at all.
Thermal protein unfolding, or thermal xe2x80x9cshift,xe2x80x9d assays have been used to determine whether a given ligand binds to a target receptor protein. In a physical thermal shift assay, a change in a biophysical parameter of a protein is monitored as a function of increasing temperature. For example, in calorimetric studies, the physical parameter measured is the change in heat capacity as a protein undergoes temperature induced unfolding transitions. Differential scanning calorimetry has been used to measure the affinity of a panel of azobenzene ligands for streptavidin (Weber, P. et al., J. Am. Chem. Soc. 16:2717-2724 (1994)). Titration calorimetry has been used to determine the binding constant of a ligand for a target protein (Brandts, J. et al., American Laboratory 22:30-41 (1990)). The calorimetric approach, however, requires that the researcher have access to a calorimetric device. In addition, calorimetric technologies do not lend themselves to the high throughput screening of combinatorial libraries, three thermal scans per day are routine. Like calorimetric technologies, spectral technologies have been used to monitor temperature induced protein unfolding (Bouvier, M. et al., Science 265:398-402 (1994); Chavan, A. J. et al., Biochemistry 33:7193-7202 (1994); Morton, A. et al., Biochemistry 1995:8564-8575 (1995)).
The single sample heating and assay configuration, as conventionally performed, has impeded the application of thermal shift technologies to high throughput screening of combinatorial libraries. Thus, there is a need for a thermal shift technology which can be used to screen combinatorial libraries, can be used to identify and rank lead compounds, and is applicable to all receptor proteins.
Thermal shift assays have been used to determine whether a ligand binds to DNA. Calorimetric, absorbance, circular dichroism, and fluorescence technologies have been used (Pilch, D. S. et al., Proc. Natl. Acad. Sci. U.S.A. 91:9332-9336 (1994); Lee, M. et al., J. Med. Chem. 36:863-870 (1993); Butour, J.-L. et al., Eur. J. Biochem. 202:975-980 (1991); Barcelo, F. et al., Chem. Biol. Interactions 74:315-324 (1990)). As used conventionally, however, these technologies have impeded the high throughput screening of nucleic acid receptors for lead compounds which bind with high affinity. Thus, there is a need for a thermal shift technology which can be used to identify and rank the affinities of lead compounds which bind to DNA sequences of interest.
When bacterial cells are used to overexpress exogenous proteins, the recombinant protein is often sequestered in bacterial cell inclusion bodies. For the recombinant protein to be useful, it must be purified from the inclusion bodies. During the purification process, the recombinant protein is denatured and must then be renatured. It is impossible to predict the renaturation conditions that will facilitate and optimize proper refolding of a given recombinant protein. Usually, a number of renaturing conditions must be tried before a satisfactory set of conditions is discovered. In a study by Tachibana et al., each of four disulfide bonds were singly removed, by site-directed mutagenesis, from hen lysozyme (Tachibanaetal, Biochemistry 33:15008-15016 (1994)). The mutant genes were expressed in bacterial cells and the recombinant proteins were isolated from inclusion bodies. Each of the isolated proteins were renatured under different temperatures and glycerol concentrations. The efficacy of protein refolding was assessed in a bacteriolytic assay in which bacteriolytic activity was measured as a function of renaturing temperature. The thermal stability of each protein was studied using a physical thermal shift assay. In this study, however, only one sample reaction was heated and assayed at a time. The single sample heating and assay configuration prevents the application of thermal shift technologies to high throughput screening of a multiplicity of protein refolding conditions. Thus, there is a need for a thermal shift technology which can be used to rank the efficacies of various protein refolding conditions.
Over the past four decades, X-ray crystallography and the resulting atomic models of proteins and nucleic acids have contributed greatly to an understanding of structural, molecular, and chemical aspects of biological phenomena. However, crystallographic analysis remains difficult because there are not straightforward methodologies for obtaining X-ray quality protein crystals. Conventional methods cannot be used quickly to identify crystallization conditions that have highest probability of promoting crystallization (Garavito, R. M. et al., J. Bioenergtics and Biomembranes 28:13-27 (1996)). Even the use of factorial design experiments and successive automated grid searches (Cox, M. J., and Weber, P. C., J. Appl. Cryst. 20:366-373 (1987); Cox, M. J., and Weber, P. C., J. Crystal Growth 90:318-324 (1988)) do not facilitate rapid, high throughput screening of biochemical conditions that promote the crystallization of X-ray quality protein crystals. Moreover, different proteins are expected to require different conditions for protein crystallization, just as has been the experience for their folding (McPherson, A., In: Preparation and Analysis of Protein Crystals, Wiley Interscience, New York, (1982)). Conventional methods of determining crystallization conditions are cumbersome, slow, and labor intensive. Thus, there is a need for a rapid, high throughput technology which can be used to rank the efficacies of protein crystallization conditions.
Rapid, high throughput screening of combinatorial molecules or biochemical conditions that stabilize target proteins in thermal shift assays would be facilitated by the simultaneous heating of many samples. To date, however, thermal shift assays have not been performed that way. Instead, the conventional approach to performing thermal shift assays has been to heat and assay only one sample at a time. That is, researchers conventionally 1) heat a sample to a desired temperature in a heating apparatus; 2) assay a physical change, such as absorption of light or change in secondary, tertiary, or quaternary protein structure; 3) heat the samples to the next highest desired temperature; 4) assay for a physical change; and 5) continue this process repeatedly until the sample has been assayed at the highest desired temperature.
This conventional approach is disadvantageous for at least two reasons. First, this approach is labor intensive. Second, this approach limits the speed with which thermal shift screening assays can be performed and thereby precludes rapid, high-throughput screening of combinatorial molecules binding to a target receptor and biochemical conditions that stabilize target proteins.
Fluorescent molecules whose spectra or quantum yields are sensitive to their environments are valuable in the study of heterogeneous media, organized media and biological media and many fluorescent dyes have been developed for these applications. However, many dyes either have short absorption and emission wavelengths (potentially causing high background due to the auto fluorescence of samples), low extinction coefficients, low quantum yields, or small Stokes shifts.
Rapid, high-throughput screening using fluorescence methodologies would also be facilitated by the use of fluorescence probe molecules that fluoresce at wavelengths longer than fluorescence molecules such as 1-anilinonaph-thalene-8-sulfonate. That is because many molecules in compound and combinatorial libraries fluoresce at the same wavelengths at which fluorescence probe molecules fluoresce. In addition, plastic microplates used in high-throughput screening assays may also fluoresce at the same wavelengths at which fluorescence probe molecules fluoresce.
Thus, there is a need for a rapid, high-throughput, fluorescence screening procedure in which fluorescence readings are taken at wavelengths longer than fluorescence molecules such as 8-anilinonaph-thalene-8-sulfonate.
The present invention provides a method for ranking the affinity of each of a multiplicity of different molecules for a target molecule which is capable of unfolding due to a thermal change, the method comprising (a) contacting the target molecule with one molecule of a multiplicity of different molecules, in the presence of a 5-(4xe2x80x3-dimethylaminophenyl)-2-(4xe2x80x2-phenyl)oxazole derivative dye, in each of a multiplicity of containers; (b) simultaneously heating the multiplicity of containers; (c) measuring the fluorescence in each of the containers; (d) generating thermal unfolding information for the target molecule as a function of temperature for each of the containers; (e) comparing the thermal unfolding information obtained for each of the containers to (i) the thermal unfolding information obtained for each of the other containers, and (ii) the thermal unfolding information obtained for the target molecule in the absence of any of the molecules in the multiplicity of different molecules; and (f) ranking the affinities of each of the molecules according to the difference in the thermal unfolding information between the target molecule in each of the containers and the target molecule in the absence of any of the molecules in the multiplicity of different molecules.
The present invention also provides a multi-variable method for ranking the affinity of a combination of two or more of a multiplicity of different molecules for a target molecule which is capable of unfolding due to a thermal change, the method comprising: (a) contacting the target molecule with a combination of two or more different molecules of the multiplicity of different molecules, in the presence of 5-(4xe2x80x3-dimethylaminophenyl)-2-(4xe2x80x2-phenyl)oxazole derivative dye, in each of a multiplicity of containers; (b) simultaneously heating the multiplicity of containers; (c) measuring in the fluorescence in each of the containers; (d) generating thermal unfolding information for the target molecule as a function of temperature in each of the containers; (e) comparing the thermal unfolding information obtained for each of the containers to (i) the thermal unfolding information obtained for each of the other containers, and (ii) the thermal unfolding information obtained for the target molecule in the absence of any of the two or more different molecules; and (f) ranking the affinities of the combinations of the two or more of the multiplicity of different molecules according to the difference in the thermal unfolding information between the target molecule in each of the containers and the thermal unfolding information obtained for the target molecule in the absence of any of the molecules in the multiplicity of different molecules.
The present invention also provides a method for assaying a collection of a multiplicity of different molecules for a molecule which binds to a target molecule which is capable of unfolding due to a thermal change, the method comprising: (a) contacting the target molecule with a collection of at least two molecules of the multiplicity of different molecules, in the presence of a 5-(4xe2x80x3-dimethylaminophenyl)-2-(4xe2x80x2-phenyl)oxazole derivative dye, in each of a multiplicity of containers; (b) simultaneously heating the multiplicity of containers; (c) measuring the fluorescence in each of the containers; (d) generating thermal unfolding information for the target molecule as a function of temperature for each of the containers; (e) comparing the thermal unfolding information obtained for each of the containers to (i) the thermal unfolding information obtained for each of the other containers, and (ii) the thermal unfolding information obtained for the target molecule in the absence of any of the multiplicity of different molecules; and (f) ranking the affinities of the collections of different molecules according to the difference in the thermal unfolding information between the target molecule in each of the containers and the thermal unfolding information obtained for the target molecule in the absence of any of the molecules in the multiplicity of different molecules; (g) selecting the collection of different molecules which contains a molecule with affinity for the target molecule; (h) dividing the selected collection into smaller collections of molecules in each of a multiplicity of containers; and (i) repeating the above steps until a single molecule, from the multiplicity of different molecules, is identified.
The present invention also provides a multi-variable method for ranking the efficacy of one or more of a multiplicity of different biochemical conditions for stabilizing a target molecule which is capable of unfolding due to a thermal change, the method comprising: (a) contacting the target molecule with one or more of the multiplicity of biochemical conditions, in the presence of a 5-(4xe2x80x3-dimethylaminophenyl)-2-(4xe2x80x2-phenyl)oxazole derivative dye, in each of a multiplicity of containers; (b) simultaneously heating the multiplicity of containers; (c) measuring the fluorescence in each of the containers; (d) generating thermal unfolding information for the target molecule as a function of temperature for each of the containers; (e) comparing the thermal unfolding information obtained for each of the containers to (i) the thermal unfolding information obtained for each of the other containers, and (ii) the thermal unfolding information obtained for the target molecule under a reference set of biochemical conditions; and (f) ranking the efficacies of each of the biochemical conditions for each of the containers according to the difference in the thermal unfolding information between the target molecule for each of the containers and the target molecule under the reference set of biochemical conditions.
The present invention also provides a multi-variable method for optimizing the shelf life of a target molecule which is capable of unfolding due to a thermal change, the method comprising: (a) contacting the target molecule with one or more of a multiplicity of different molecules or different biochemical conditions, in the presence of a 5-(4xe2x80x3-dimethylaminophenyl)-2-(4xe2x80x2-phenyl)oxazole derivative dye, in each of a multiplicity of containers; (b) simultaneously heating the multiplicity of containers; (c) measuring the fluorescence in each of the containers; (d) generating thermal unfolding information for the target molecule as a function of temperature for each of the containers; (e) comparing the thermal unfolding information obtained for each of the containers to (i) the thermal unfolding information obtained for each of the other containers, and (ii) the thermal unfolding information obtained for the target molecule under a reference set of biochemical conditions; and (f) ranking the efficacies of each of the biochemical conditions for each of the containers according to the difference in the thermal unfolding information between the target molecule for each of the containers and the target molecule under the reference set of biochemical conditions.
The present invention also provides a multi-variable method for ranking the efficacies of one or more of a multiplicity of different biochemical conditions to facilitate the refolding or renaturation of a sample of a denatured or unfolded protein, the method comprising: (a) placing one of the refolded protein samples, in the presence of a 5-(4xe2x80x3-dimethylaminophenyl)-2-(4xe2x80x2-phenyl)oxazole derivative dye, in each of a multiplicity of containers, wherein each of the refolded protein samples has been previously refolded or renatured according to one or more of the multiplicity of conditions; (b) simultaneously heating the multiplicity of containers; (c) measuring the fluorescence in each of the containers; (d) generating thermal unfolding information for the target molecule as a function of temperature for each of the containers; (e) comparing the thermal unfolding information obtained for each of the containers to (i) the thermal unfolding information obtained for each of the other containers, and (ii) the thermal unfolding information obtained for the target molecule under a reference set of biochemical conditions; and (f) ranking the efficacies of each of the biochemical conditions for each of the containers according to the difference in the thermal unfolding information between the target molecule for each of the containers and the target molecule under the reference set of biochemical conditions.
The present invention also provides a multi-variable method for ranking the efficacies of one or more of a multiplicity of different biochemical conditions to facilitate the refolding or renaturation of a sample of a denatured unfolded protein, the method comprising: (a) determining one or more combinations of a multiplicity of different conditions which promote protein stabililty, incubating denatured protein under the one or more combinations of biochemical conditions that were identified as promoting protein stabilization; (b) assessing folded protein yield; (c) ranking the efficacies of the multiplicity of different refolding conditions according to folded protein yield; and (d) repeating these steps until a combination of biochemical conditions that promote optimal protein folding are identified.
The present invention also provides a multi-variable method for ranking the efficacy of one or more of a multiplicity of different biochemical conditions for facilitating the crystallization of a protein which is capable of unfolding due to a thermal change, the method comprising: (a) contacting the protein with one or more of the multiplicity of different biochemical conditions, in the presence of a 5-(4xe2x80x3-dimethylaminophenyl)-2-(4xe2x80x2-phenyl)oxazole derivative dye, in each of a multiplicity of containers; (b) simultaneously heating the multiplicity of containers; (c) measuring the fluorescence in each of the containers; (d) generating thermal unfolding information for the target molecule as a function of temperature for each of the containers; (e) comparing the thermal unfolding information obtained for each of the containers to (i) the thermal unfolding information obtained for each of the other containers, and (ii) the thermal unfolding information obtained for the target molecule under a reference set of biochemical conditions; and ranking the efficacies of each of the biochemical conditions for each of the containers according to the difference in the thermal unfolding information between the target molecule for each of the containers and the target molecule under the reference set of biochemical conditions.
Optimization of protein stability, ligand binding, protein folding, and protein crystallization are multi-variable events. Multi-variable optimization problems require large numbers of parallel experiments to collect as much data as possible in order to determine which variables influence a favorable response. For example, multi-variable optimization problems require large numbers of parallel experiments to collect as much data as possible in order to determine which variables influence protein stabililty. In this regard, both protein crystallization and quantitative structure activity relationship analyses have greatly benefited from mass screening protocols that employ matrix arrays of incremental changes in biochemical or chemical composition. Thus, in much the same way that quantitative structure activity relationships are constructed to relate variations of chemical finctional groups on ligands to their effect on binding affinity to a given therapeutic receptor, the methods and apparatus of the present invention facilitate the construction of a quantitative model that relates different biochemical conditions to experimentally measured protein stability, ligand specificity, folded protein yield, and crystallized protein yield.
Using the fluorescence microplate thermal shift assay, one can determine one or more biochemical conditions that have an additive effect on protein stability. Once a set of biochemical conditions that facilitate an increase in protein stability have been identified using the thermal shift assay, the same set of conditions can be used in protein folding experiments with recombinant protein. If the conditions that promote protein stability in the thermal shift assay correlate with conditions that promote folding of recombinant protein, conditions can be further optimized by performing additional thermal shift assays until a combination of stabilizing conditions that result in further increase protein stability are identified. Recombinant protein is then folded under those conditions. This process is repeated until optimal folding conditions are identified.
The present invention offers a number of advantages over previous technologies that are employed to optimize multi-variable events such as protein stabilization, ligand binding, protein folding, and protein crystallization. Foremost among these advantages is that the present invention facilitates high throughput screening. The use of 5-(4xe2x80x3-dimethylaminophenyl)-2-(4xe2x80x2-phenyl)oxazole derivative dyes to practice the microplate thermal shift assay of the present invention affords increased assay sensitivity and increased assay throughput, because these dyes have long emission wavelengths, high extinction coefficients, high quantum yields, and large Stokes shifts.
Further, the methods of the present invention offers a number of advantages over previous technologies that are employed to screen combinatorial libraries. Foremost among these advantages is that the present invention facilitates high throughput screening of combinatorial libraries for lead compounds. Many current library screening technologies simply indicate whether a ligand binds to a receptor or not. In that case, no quantitative information is provided. No information about the relative binding affinities of a series of ligands is provided. In contrast, the present invention facilitates the ranking of a series of compounds for their relative affinities for a target receptor. With this information in hand, a structure-activity relationship can be developed for a set of compounds. The ease, reproducibility, and speed of using ligand-dependent changes in midpoint unfolding temperature (Tm) to rank relative binding affinities makes the present invention a powerful tool in the drug discovery process.
Typically, the conventional kinetic screening approach requires at least six additional well assays at six different concentrations of inhibitor to determine a Ki. Using the present invention, throughput is enhanced xcx9c6 fold over the enzyme based assays because one complete binding experiment can be performed in each well of a multiwell microplate. The kinetic screening approached are even further limited by the usual compromise between dilution and signal detection, which usually occurs at a protein concentration of about 1 nM. In this regard, the calorimetric approaches, either differential scanning calorimetry or isothermal titrating calorimetry, are at an even worse disadvantage since they are limited to solitary binding experiments, usually 1 per hour. In contrast, the present invention affords a wide dynamic range of measurable binding affinities, from xcx9c10xe2x88x924 to 10xe2x88x9215 M, in a single well.
A very important advantage of the present invention is that it can be applied universally to any receptor that is a drug target. Thus, it is not necessary to invent a new assay every time a new receptor becomes available for testing. When the receptor under study is an enzyme, researchers can determine the rank order of affinity of a series of compounds more quickly and more easily than they can using conventional kinetic methods. In addition, researchers can detect ligand binding to an enzyme, regardless of whether binding occurs at the active site, at an allosteric cofactor binding site, or at a receptor subunit interface. The present invention is equally applicable to non-enzyme receptors, such as proteins and nucleic acids.
Further features and advantages of the present invention are described in detail below with reference to the accompanying drawings.